Metastatic breast cancer in women is presently treated with a variety of modalities of therapy. Autologous bone marrow transplantation (ABMT) prior to chemotherapy, while potentially effective, suffers from the problem that it is difficult to identify and remove all metastatic cells from the bone marrow (BM). Metastatic cells in peripheral blood (PB) are also difficult to identify and remove. An important aspect of this problem is to identify and characterize the metastatic cells present before and after cleansing techniques. These metastatic cells are so rare that they are undetectable by most means. What properties of the rare metastatic cells significantly differ from those of the primary lesion? Can these differences be exploited to help remove them by other techniques directly from peripheral blood and/or bone marrow? If successful, ABMT when combined with chemotherapy would be an important and effective systemic therapy. Using new special high-speed (greater than 100,000 cells/sec) flow cytometric detection of ultra-rare (less than 10-6 rare cell frequencies) it should now be possible to detect and isolate rare metastatic breast cells in BM and in PB at frequencies below one metastatic cell per million normal cells. Special high-speed sorting techniques will be used with subsequent image analysis and PCR analysis for subsequent molecular characterization of single or small numbers of these rare metastatic cells. Attempts will be made to purge them from BM. Such immunocytochemical and molecular characterizations of these cells could provide improved understanding of the underlying causes of metastatic breast cancer and may suggest new therapies. Rare metastatic cells in PB and BM will be identified on the basis of multiple positive selection markers including (but not limited to) epithelial and breast cancer cell surface markers PHM-5, 2G3, 9C6, 741F8, and/or intracellular markers GCDFP-15 and cytokeratins AE1/AE3 and multiple negative selection markers. No single marker has thus far proved unique either for positive or negative selection, but there may well exist a combination of such positive and negative markers sufficient to perform either a single-cell multi-tube sort or an impure sort with subsequent further processing by image analysis with additional markers and cloning either for growth or for """"""""cookie-cutter"""""""" re-sorting for PCR characterization with one or more molecular markers. Comparisons of molecular characterizations of metastatic cells with those in the primary lesion will be performed using PCR sequences for a variety of genes frequently amplified or over- expressed in metastatic breast cancer cells including c-erbB2, c-myc, int- 2 and mts1.
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